OMIP-098: A 26 parameter, 24 color flow cytometry panel for human memory NK cell phenotyping

Abstract

This 26-parameter flow cytometry panel has been developed and optimized to analyze NK cell phenotype, using cryopreserved peripheral blood mononuclear cells (PBMCs) from people living with and without human immunodeficiency virus (PLWH, PWOH). Our panel is designed for the analysis of several parameters of total NK cells and memory NK cell subsets including markers of maturation, activation, and proliferation, as well as activating and inhibitory receptors. Other tissues have not been tested (Table 1).

Figure 1:

Example of staining, gating, and phenotypic analysis of memory NK cells. A) Identification of NK cells from a representative example of a donor without HIV. First, we verified that there were no irregularities in the acquisition over time (SSC-A vs. Time). From single cells (SSC-H vs. SSC-A) we selected live (Aqua⁻) small lymphocytes (SSC-A vs FSC-A). After exclusion of the lineage-negative cells expressing either CD3/CD19/CD33, total NK cells were identified by the expression of the markers CD56 and CD16. Within the total NK cells, we identified 3 NK cell subsets based on the expression of CD56: early CD56^bright, mature CD56^dim, and the dysfunctional CD56^neg NK cells. We also characterized within the total NK cells the CD57⁺ NKG2C⁺ adaptive memory NK cells and the FcεRIγ⁻ NKG2C⁺ ΔgNK memory cells. B) As the CD57⁺ NKG2C⁺ adaptive memory NK cells and the FcεRIγ⁻ NKG2C⁺ ΔgNK memory cells are overlapping populations, from the total NK cells we define NKG2C+ cells as memory NK cells and subsequently gate the different memory subsets based on the expression of CD57 and FcεRIγ as the following: NKG2C+ CD57+ FcεRIγ-, NKG2C+ CD57+ FcεRIγ+, NKG2C+ CD57- FcεRIγ+, NKG2C+ CD57- FcεRIγ−. C) Representative dot plots of the expression of the parameters included in our panel on total NK cells (i) and NKG2C+ memory NK cells (ii).